The capacity of lipid molecules in cell membranes to separate into multiple liquid phases, forming so- called lipid rafts, has in the past decade been identified as an important physiological process. Lipid rafts are a control element in the cell membrane, and they take part in numerous molecular pathways with implications for human health, including signal transduction and viral entry. In vitro models of the cell membrane built from synthetic bilayers have played an important role in elucidating the fundamental mechanisms underlying raft formation. Existing artificial bilayer models, however, lack some properties that are inherent to the cell plasma membrane and that are likely important in reproducing the physiological behavior of lipids. These properties include the mechanical attachment of the bilayer to an underlying polymeric cytoskeleton and the compositional asymmetry of the cell membrane. This proposal calls for the fabrication of novel artificial bilayer constructs that will better mimic the structure of the cell plasma membrane and serve as research platforms for investigating lipid raft behavior. This will be accomplished by building vesicular bilayer structures (so called giant unilamellar vesicles, or GUVs) that are filled with a polymer hydrogel. This hydrogel will serve as a biomimetic cytoskeleton, and the membrane will be physically anchored to it via chemical conjugation. One major shortcoming of existing artificial bilayer models of the cell membrane is their inability to properly recapitulate the nanometer scale of lipid rafts found in actual cells, producing instead micrometer-sized lipid domains. There is significant evidence that the size of rafts in cells is limited by the mechanical attachment of the membrane to the cytoskeleton. Building a biomimetic cytoskeleton will allow for precise control over the nature and density of membrane-cytoskeleton attachments and therefore a detailed investigation of the relationship between cytoskeletal attachment and raft size. It will also provide a versatile research platform that can be used to investigate a wide variety of lipid structure-related questions. Investigation of lipid structures at the nanoscale requires the development of analytical techniques that can address these tiny structures. This proposal outlines a set of techniques based on total internal reflection fluorescence microscopy and Fvrster resonance energy transfer that will allow for the detection and evaluation of nanoscale rafts with spatial and temporal resolution. Also proposed is a microfluidic technology for assembling bilayers on GUVs in a layer-by-layer fashion, allowing for the composition of each layer to be controlled and facilitating the fabrication of asymmetric bilayers like those that compose the plasma membrane. Together with the hydrogel cytoskeleton, this technology will allow for a new type of artificial cell that mimics accurately most important properties of the eukaryotic plasma membrane. PUBLIC HEALTH RELEVANCE: Lipid nanostructures in cell membranes help control how cells interact with their environments and are therefore central actors in many disease states, including type-2 diabetes and viral infection. While synthetic lipid bilayers modeling the cell membrane have been important tools for elucidating the molecular mechanisms that underlie lipid structure formation, these systems fail to reproduce important properties of real cell membranes. The new artificial cell constructs proposed here mimic both the cytoskeletal attachment and compositional asymmetry found in cell membranes, allowing them to serve as research platforms for understanding how lipid nanostructures behave and how novel therapeutic approaches can alter lipid- mediated processes.